Dataset Information

MudPIT analyses of the protein succination after fumarate treatment of whole cell extracts from Hereditary Leiomyomatosis and Renal Cell Cancer (HLRCC) fumarate hydratase-deficient (FH-/-) cells

ABSTRACT: In brief, TCA-precipitated proteins from whole cell extracts treated with fumarate were urea-denatured, reduced, alkylated, and digested with endoproteinase LysC followed by trypsin. The peptide mixtures were loaded onto microcapillary fused silica columns (100um i.d.), packed with C18 reverse phase (Aqua; Phenomenx), SCX (Luna; Phenomenex) and C18-RP, placed in-line with an Agilent 11000 quaternary pump, and analyzed by a 10-step MudPIT on Orbitrap Velos Pro or Elite. Full MS spectra were recorded on the eluting peptides over a 400 to 1600 m/z range in the Orbitrap at 60K resolution, followed by fragmentation in the ion trap (at 35% collision energy) on the first to fifteenth most intense ions selected from the full MS spectrum. MS/MS datasets searched using ProLuCID (Xu et al., 2015) with a peptide mass tolerance of 10 ppm and 500 ppm for fragment ions. Trypsin specificity was imposed on both ends of candidate peptides during the search against a protein database combining 36,628 human proteins (NCBI 2016-06-10 release), as well as 193 usual contaminants such as human keratins, IgGs and proteolytic enzymes. To estimate false discovery rates (FDR), each protein sequence was randomized (keeping the same amino acid composition and length) and the resulting "shuffled" sequences were added to the database, for a total search space of 73,642 amino acid sequences. Masses of 57.0215 Da and 116.0112 Da were differentially added to cysteine residues to account for alkylation by CAM and succination, respectively, while 15.9949 Da were differentially added to methionine residues. Peptide/spectrum matches were sorted and selected using DTASelect with the following criteria set: spectra/peptide matches were retained only if they had a DeltCn of at least 0.8, and minimum XCorr of 1.8 for singly, 2.0 for doubly, and 3.0 for triply charged spectra. Additionally, the peptides had to be minimum 7 amino acids in length and fully tryptic.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Laurence Florens

PROVIDER: MSV000082160 | MassIVE | Tue Mar 13 08:55:00 GMT 2018

SECONDARY ACCESSION(S): PXD009202

REPOSITORIES: MassIVE

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Publications

A chemoproteomic portrait of the oncometabolite fumarate.

Kulkarni Rhushikesh A RA Bak Daniel W DW Wei Darmood D Bergholtz Sarah E SE Briney Chloe A CA Shrimp Jonathan H JH Alpsoy Aktan A Thorpe Abigail L AL Bavari Arissa E AE Crooks Daniel R DR Levy Michaella M Florens Laurence L Washburn Michael P MP Frizzell Norma N Dykhuizen Emily C EC Weerapana Eranthie E Linehan W Marston WM Meier Jordan L JL

Nature chemical biology 20190204 4

Hereditary cancer disorders often provide an important window into novel mechanisms supporting tumor growth. Understanding these mechanisms thus represents a vital goal. Toward this goal, here we report a chemoproteomic map of fumarate, a covalent oncometabolite whose accumulation marks the genetic cancer syndrome hereditary leiomyomatosis and renal cell carcinoma (HLRCC). We applied a fumarate-competitive chemoproteomic probe in concert with LC-MS/MS to discover new cysteines sensitive to fumar ...[more]

PMID: 30718813

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Dataset Information

MudPIT analyses of the protein succination after fumarate treatment of whole cell extracts from Hereditary Leiomyomatosis and Renal Cell Cancer (HLRCC) fumarate hydratase-deficient (FH-/-) cells

Publications

A chemoproteomic portrait of the oncometabolite fumarate.

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